Neuroepithelial Bodies of Pulmonary Airways Serve as a Reservoir of Progenitor Cells Capable of Epithelial Regeneration
2000; Elsevier BV; Volume: 156; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64727-x
ISSN1525-2191
AutoresSusan D. Reynolds, Adam Giangreco, John H. Power, Barry R. Stripp,
Tópico(s)Neuroscience of respiration and sleep
ResumoRemodeling of the conducting airway epithelium is a common finding in the chronically injured lung and has been associated with increased risk for developing lung cancer. Pulmonary neuroendocrine cells and clusters of these cells termed neuroepithelial bodies (NEBs) play a central role in each of these processes. We previously developed an adult mouse model of airway injury and repair in which epithelial regeneration after naphthalene-induced Clara cell ablation occurred preferentially at airway branch points and gave rise to nascent Clara cells. Continued repair was accompanied by NEB hyperplasia. We now provide the following evidence that the NEB microenvironment serves as a source of airway progenitor cells that contribute to focal regeneration of the airway epithelium: 1) nascent Clara cells and NEBs localize to the same spatial domain; 2) within NEB, both Clara cell secretory protein- and calcitonin gene-related peptide-immunopositive cells are proliferative; 3) the NEB microenvironment of both the steady-state and repairing lung includes cells that are dually immunopositive for Clara cell secretory protein and calcitonin gene-related peptide, which were previously identified only within the embryonic lung; and 4) NEBs harbor variant Clara cells deficient in cytochrome p450 2F2-immunoreactive protein. These data suggest that the NEB microenvironment is a reservoir of pollutant-resistant progenitor cells responsive to depletion of an abundant airway progenitor such as the Clara cell. Remodeling of the conducting airway epithelium is a common finding in the chronically injured lung and has been associated with increased risk for developing lung cancer. Pulmonary neuroendocrine cells and clusters of these cells termed neuroepithelial bodies (NEBs) play a central role in each of these processes. We previously developed an adult mouse model of airway injury and repair in which epithelial regeneration after naphthalene-induced Clara cell ablation occurred preferentially at airway branch points and gave rise to nascent Clara cells. Continued repair was accompanied by NEB hyperplasia. We now provide the following evidence that the NEB microenvironment serves as a source of airway progenitor cells that contribute to focal regeneration of the airway epithelium: 1) nascent Clara cells and NEBs localize to the same spatial domain; 2) within NEB, both Clara cell secretory protein- and calcitonin gene-related peptide-immunopositive cells are proliferative; 3) the NEB microenvironment of both the steady-state and repairing lung includes cells that are dually immunopositive for Clara cell secretory protein and calcitonin gene-related peptide, which were previously identified only within the embryonic lung; and 4) NEBs harbor variant Clara cells deficient in cytochrome p450 2F2-immunoreactive protein. These data suggest that the NEB microenvironment is a reservoir of pollutant-resistant progenitor cells responsive to depletion of an abundant airway progenitor such as the Clara cell. Conducting airways of the lung are lined by a variety of specialized epithelial cells that fulfill critical functions necessary for the viability of air-breathing organisms. These functions include clearance and, in some cases, metabolism of inhaled environmental agents, regulation of lung fluid balance, and renewal of the epithelium after injury.1Rennard SI, Beckmann JD, Robbins RA: Biology of airway epithelial cells. The Lung: Scientific Foundations Edited by RG Crystal, JB West. New York, Raven Press, pp 157–167Google Scholar Altered airway epithelial cell composition that is associated with acute or chronic lung injury has the potential to significantly impact these functions.2Carter PM Heinly TL Yates SW Lieberman PL Asthma: the irreversible airways disease.J Invest Allergol Clin Immunol. 1997; 7: 566-571PubMed Google Scholar, 3Frampton MW Morrow PE Torres A Voter KZ Whitin JC Cox C Speers DM Tsai Y Utell MJ Effects of ozone on normal and potentially sensitive human subjects. Part II. Airway inflammation and responsiveness to ozone in nonsmokers and smokers.Res Rep Health Effects Inst. 1997; 78: 39-72PubMed Google Scholar Among the many agents that cause lung injury, pneumotoxic environmental pollutants contribute significantly to morbidity and mortality in a significant fraction of the human population.4Bates DV The effects of air pollution on children.Environ Health Perspect. 1995; 103: 49-53Crossref PubMed Scopus (122) Google Scholar, 5Leikauf GD Kline S Albert RE Baxter CS Bernstein DI Buncher CR Evaluation of a possible association of urban air toxics and asthma.Environ Health Perspect. 1995; 102: 253-271Google Scholar Mechanisms by which these pollutants exert their effects involve cellular injury leading to dysregulation of growth and/or differentiation of epithelial cells. Associated changes in airway epithelial cell composition often involve transient or persistent hyperplasias such as squamous cell, neuroendocrine cell, basal cell, and secretory cell hyperplasia. These changes to the conducting airway epithelium increase the risk of developing either small-cell or non-small-cell lung cancers in smokers6Nakanishi K Hiroi S Kawai T Suzuki M Torikata C Argyrophilic nucleolar-organizer region counts and DNA status in bronchioloalveolar epithelial hyperplasia and adenocarcinona of the lung.Hum Pathol. 1998; 29: 235-239Abstract Full Text PDF PubMed Scopus (20) Google Scholar, 7Betticher DC Heighway J Thatcher N Hasleton PS Abnormal expression of CCND1 and RB1 in resection margin epithelia of lung cancer patients.Br J Cancer. 1997; 75: 1761-1768Crossref PubMed Scopus (45) Google Scholar, 8Smith AL Hung J Walker L Rogers TE Vuitch F Lee E Gazear AF Extensive areas of aneuploidy are present in the respiratory epithelium of lung cancer patients.Br J Cancer. 1996; 73: 203-209Crossref PubMed Scopus (70) Google Scholar, 9Agapitos E Molo F Tomatis L Katsouyanni K Lipworth L Delsedime L Kalandidi A Karakatsani A Riboli E Saracci R Trichopoulos D Epithelial, possibly precancerous, lesions of the lung in relation to smoking, passive smoking, and socio-demographic variables.Scan J Soc Med. 1996; 24: 259-263PubMed Google Scholar, 10Crowell RE Gilliland FD Temes RT Harms HJ Neft RE Heaphy E Auckley DH Crooks LA Jordan SW Samet JM Lechner JF Belinsky SA Detection of trisomy 7 in nonmalignant bronchial epithelium from lung cancer patients and individuals at risk for lung cancer.Cancer Epidemiol Biomarkers Prevent. 1996; 5: 631-637PubMed Google Scholar, 11Trichopoulos D Mollo F Tomatis L Agapitos E Delsedime L Zavitsanos X Kalandidi A Katsouyanni K Riboli E Saracci R Active and passive smoking and pathological indicators of lung cancer risk in an autopsy study.J Am Med Assoc. 1992; 268: 1697-1701Crossref Scopus (51) Google Scholar and in nonsmokers.12Alavanja MC Brownson RC Boice JD Hock E Preexisting lung disease and lung cancer among nonsmoking women.Am J Epidemiol. 1992; 136: 623-632PubMed Google Scholar, 13Osann KE Lung cancer in women: the importance of smoking, family history of cancer and medical history of respiratory disease.Cancer Res. 1991; 51: 4893-4897PubMed Google Scholar Both pulmonary neuroendocrine (PNE) cells and Clara cells have been proposed as progenitors for the genesis of small-cell and non-small-cell lung cancers. Clara cells represent the most abundant secretory cell type of distal airways in the human lung and of both proximal and distal airways in the lungs of rodents and rabbits.14Plopper CG Mariassy AT Wilson DW Alley JL Nishio SJ Nettesheim P Comparison of nonciliated trachial epithelial cells in six mammalian species: ultrastructure and population densities.Exp Lung Res. 1983; 5: 281-294Crossref PubMed Scopus (106) Google Scholar In addition to their secretory function, Clara cells are a major site for cytochrome p450-mediated oxidation of both endogenous and xenobiotic lipophilic compounds in the lung,15Plopper CG Dungworth DL Structure, function, cell injury, and cell renewal of bronchiolar, and alveolar epithelium.in: McDowell EM Lung Carcinoma. Churchill Livingstone, London1987: 29-44Google Scholar, 16Devereux TR Donim BA Philpot RM Xenobiotic metabolism by isolated pulmonary cells.Pharmacol Ther. 1989; 41: 243-256Crossref PubMed Scopus (90) Google Scholar and they serve as the principal source of progenitor cells for repopulation of airways after injury by inhaled oxidant pollutants.17Evans MJ Johnson LV Stephens RJ Freeman G Renewal of the terminal bronchiolar epithelium in the rat following exposure to NO2 or O3.Lab Invest. 1976; 35: 246-257PubMed Google Scholar A morphological derivative of the Clara cell, referred to as a type-A cell, enters the cell cycle after oxidant-induced airway injury.18Evans MJ Cabral-Anderson LJ Freeman G Role of the Clara cell in renewal of the bronchiolar epithelium.Lab Invest. 1978; 38: 648-653PubMed Google Scholar Type-A cells lack the differentiated ultrastructural features, such as smooth endoplasmic reticulum and secretory granules, that are characteristic of mature Clara cells.17Evans MJ Johnson LV Stephens RJ Freeman G Renewal of the terminal bronchiolar epithelium in the rat following exposure to NO2 or O3.Lab Invest. 1976; 35: 246-257PubMed Google Scholar, 18Evans MJ Cabral-Anderson LJ Freeman G Role of the Clara cell in renewal of the bronchiolar epithelium.Lab Invest. 1978; 38: 648-653PubMed Google Scholar Cells with Clara-like characteristics but reduced secretory protein expression have been identified in association with preneoplastic lesions and represent a subpopulation of non-small-cell lung cancers.19Linnoila RI Mulshine JL Steinberg SM Funa K Matthews MJ Cotelingam JD Gazdar AF Neuroendocrine differentiation in endocrine and nonendocrine lung carcinomas.Am J Clin Pathol. 1988; 90: 641-652PubMed Google Scholar, 20Linnoila RI Jensen SM Steinberg SM Mulshine JL Eggleston JC Gazdar AF Peripheral airway cell marker expression in non-small cell lung carcinoma.Am J Clin Pathol. 1992; 97: 233-243Crossref PubMed Scopus (91) Google Scholar, 21linnoila RI Aisner SC Pathology of lung cancer: exercise in classification. Lung Cancer.in: Johnson BE Johnson DH Current Clinical Oncology. John Wiley & Sons, New York1994: 73-95Google Scholar The progenitor role of Clara cells makes them a likely target for deregulated growth control and neoplastic progression. In contrast, PNE cells are not considered to be a progenitor population in the adult lung and are generally thought to be terminally differentiated and mitotically inert.22Gosney JR Pulmonary neuroendocrine cell system in pediatric and adult lung disease.Microsc Res Tech. 1997; 37: 107-113Crossref PubMed Scopus (39) Google Scholar In the absence of demonstrable PNE proliferation, it has been suggested that maintenance of PNE cells in the normal lung23Linnoila RI Effects of diethylnitrosamine of lung neuroepithelial cells.Exp Lung Res. 1982; 3: 225-236Crossref PubMed Scopus (30) Google Scholar and PNE cell hyperplasia during repair24Sunday ME Willett CG Patidar K Graham SA Modulation of oncogene and tumor suppressor gene expression in a hamster model of chronic lung injury with varying degrees of pulmonary neuroendocrine cell hyperplasia.Lab Invest. 1994; 70: 875-888PubMed Google Scholar, 25Sunday ME Willett CG Induction and spontaneous regression of intense pulmonary neuroendocrine cell differentiation in a model of preneoplastic lung injury.Cancer Res. 1992; 52: 2677S-2686SPubMed Google Scholar result from proliferation and differentiation of a progenitor population with multipotent differentiation capacity. However, this notion has been challenged by the finding that repair from airway injury is associated with PNE cell hyperplasia and that proliferation contributes to this hyperplastic response.23Linnoila RI Effects of diethylnitrosamine of lung neuroepithelial cells.Exp Lung Res. 1982; 3: 225-236Crossref PubMed Scopus (30) Google Scholar, 26Ito T Ikemi Y Ohmori K Kiamura H Kanisawa M Airway epithelial cell changes in rats exposed to 0.25 ppm ozone for 20 months.Exp Toxic Pathol. 1994; 46: 1-6Crossref PubMed Scopus (16) Google Scholar, 27Stevens T McBride JT Peake JL Pinkerton KE Stripp BR Cell proliferation contributes to PNEC hyperplasia following acute airway injury in adult mice.Am J Physiol. 1997; 272: L486-L493PubMed Google Scholar It is clear that additional studies are necessary to characterize the progenitor for the PNE cell lineage and to determine whether PNE hyperplasia contributes productively to airway repair. The suggestion that pulmonary epithelial cells of the developing lung coexpress genes normally expressed by epithelial cells that are considered to be of distinct lineages raises the possibility that PNE cells may serve as a pluripotent progenitor cell.28Wuenschell CW Sunday ME Singh G Minoo P Slavkin HD Warburton D Embryonic mouse lung epithelial progenitor cells co-express immunohistochemial markers of diverse mature cell lineages.J Histochem Cytochem. 1996; 44: 113-123Crossref PubMed Scopus (100) Google Scholar, 29Wert SE Glasser SW Korfhagen TR Whitsett JA Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice.Dev Biol. 1993; 156: 426-443Crossref PubMed Scopus (275) Google Scholar In addition, studies investigating the contribution of PNE cell-derived neuropeptides in lung development suggest a role for PNE cells in regulation of epithelial cell renewal through elaboration of paracrine regulatory factors.30Cutz E Gillan JE Perrin DG Pulmonary neuroendocrine cell system: an overview of cell biology and pathology with emphasis of pediatric lung disease.in: Askin FB Langston FC Bernstein J Pulmonary Disease: Perspectives in Pediatric Pathology. vol. 18. Kager, Basel1995: 32-70Google Scholar This possibility is supported by studies in developing pulmonary airways that show an inverse relationship between epithelial mitotic index and distance from clusters of PNE cells, termed neuroepithelial bodies (NEBs)31Holt RF McNelly NA Sorokin SP Dynamics of neuroepithelial body (NEB) formation in developing hamster lung: light microscopic autoradiography after 3H-thymidine labeling in vivo.Anat Rec. 1990; 227: 340-350Crossref PubMed Scopus (46) Google Scholar and the observation that neuropeptides such as gastrin-releasing peptide (GRP) have been shown to function as potent epithelial cell mitogens and contribute to lung branching.32Sunday ME Hua J Dai HB Nusrat A Torday JS Bombesin increases fetal lung growth and maturation in utero and in organ cultures.Am J Respir Cell Mol Biol. 1990; 3: 199-205Crossref PubMed Scopus (141) Google Scholar, 33Sunday ME Choi N Spindel ER Chin WW Mark E Gastrin-releasing peptide gene expression in small cell and large cell undifferentiated lung carcinomas.Hum Pathol. 1991; 22: 1030-1039Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 34Sunday ME Hua J Reyes B Masui H Torday JS Anti-bombesin antibodies modulate fetal murine lung growth and maturation in utero and in organ cultures.Anat Rec. 1993; 236: 25-32Crossref PubMed Scopus (65) Google Scholar We and others have shown that parenteral administration of naphthalene to mice results in acute Clara cell toxicity.35Stripp BR Maxson K Mera R Singh G Plasticity of airway cell proliferation and gene expression after acute naphthalene injury.Am J Physiol. 1995; 269: L791-L799PubMed Google Scholar, 36Plopper CG Suverkropp C Morin D Nishio S Buckpitt A Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene.J Pharmacol Exp Ther. 1992; 26: 353-363Google Scholar, 37Mahvi D Bank H Harley R Morphology of a naphthalene-induced bronchiolar lesion.Am J Pathol. 1977; 86: 559-572Google Scholar Naphthalene is metabolized by cytochrome p450–2F2 (CYP-2F2) and -2B2 isoenzymes, expressed principally within Clara cells of the mouse lung, resulting in the production of the highly toxic naphthalene 1R, 2S epoxide.38Buckpitt AR Buonarati M Avey LB Chang AM Morin D Plopper C Relationship of cytochrome P450 activity to Clara cell cytotoxicity. II. Comparison of stereoselectivity of naphthalene epoxidation in lung and nasal mucosa of mouse, hamster, rat and rhesus monkey.J Pharmacol Exp Ther. 1992; 261: 364-372PubMed Google Scholar Renewal of the injured airway involves focal-cell proliferation that leads to restoration of Clara cell secretory protein (CCSP)-expressing cells and hyperplasia of calcitonin gene-related peptide (CGRP)-immunopositive (IP) PNE cells.27Stevens T McBride JT Peake JL Pinkerton KE Stripp BR Cell proliferation contributes to PNEC hyperplasia following acute airway injury in adult mice.Am J Physiol. 1997; 272: L486-L493PubMed Google Scholar, 35Stripp BR Maxson K Mera R Singh G Plasticity of airway cell proliferation and gene expression after acute naphthalene injury.Am J Physiol. 1995; 269: L791-L799PubMed Google Scholar The goal of the current study was to investigate the relationship between Clara cells and PNE cells in repairing airways of adult naphthalene-exposed mice. These studies test the hypothesis that the NEB microenvironment is critical for the maintenance of a “reserve” progenitor-cell population in the mature lung that participates in airway repair after acute airway injury involving an abundant progenitor cell (the Clara cell). We demonstrate that both Clara cells and PNE cells proliferate during airway repair and that both populations colocalize to the neuroepithelial body. Moreover, we demonstrate that the NEB microenvironment of the normal and injured lung supports the maintenance of at least two epithelial cell variants, one with a phenotype intermediate between Clara and PNE cells, and a Clara cell variant with little or no immunoreactive CYP-2F2 protein. These studies suggest a novel role for NEB in maintenance of multiple progenitor-cell populations in the mature airway. Wild-type male FVB/n mice (2–4 months old) used in this study were maintained as a specific pathogen-free in-house colony. They were allowed food and water ad libitum and maintained on a 12-hour/day light-dark cycle. Representative animals from the colony were screened quarterly using a comprehensive 16-agent serological panel, and for the absence of ecto- and endoparasites (Microbiological Associates, Rockville, MD). Naphthalene was obtained from Sigma Chemical Co. (St. Louis, MO). Naphthalene was dissolved in Mazola corn oil at a concentration of 30 mg/ml and administered intraperitoneally at 10 ml/kg body weight. One hour before sacrifice, animals were injected intraperitoneally with 2.5 mCi [3H]-thymidine/kg. Groups of three mice were exposed to 1.0 part per million (ppm) O3 for 48 or 72 hours as previously described.39Mango GW Johnston CJ Reynolds SD Finkelstein JN Plopper CG Stripp BR Clara cell secretory protein deficiency increases oxidant stress response in conducting airways.Am J Physiol. 1998; 275: L348-L356PubMed Google Scholar Control mice for naphthalene and ozone exposures received either 10 ml of corn oil/kg body weight or were exposed to filtered room air, respectively. Mice were sacrificed by intraperitoneal injection of 100 mg/kg sodium pentobarbital and were exsanguinated. Lungs were inflation fixed through a tracheal cannula at 10 cm water pressure with neutral buffered formalin for 10 minutes and immersion fixed in the same fixative overnight, followed by 24 hours in phosphate-buffered saline (PBS). The infracardiac lobe was dehydrated and embedded in paraffin. Adjacent serial 5 μm sections were cut to reveal the major axial pathway, three to five minor daughter airways, and numerous terminal bronchioles. Sections were baked at 60°C overnight, cleared with three changes of xylene, and hydrated in a graded ethanol series to water. Endogenous peroxidase activity was quenched with 3% aqueous hydrogen peroxide for 10 minutes, and the tissue was blocked with PBS/0.5. bovine serum albumin (BSA)/5% normal goat serum for 30 minutes. Rabbit anti-CCSP was obtained from G. Singh (University of Pittsburgh, Pittsburgh, PA) and used at a dilution of 1:12,000. CCSP is abundant, is expressed in both immature and mature Clara cells, and serves as a reliable immunohistochemical marker for this cell type (28 and references therein). Previous in situ hybridization analysis has demonstrated that nascent Clara cells of the regenerating naphthalene-exposed airway express CCSP mRNA.35Stripp BR Maxson K Mera R Singh G Plasticity of airway cell proliferation and gene expression after acute naphthalene injury.Am J Physiol. 1995; 269: L791-L799PubMed Google Scholar Rabbit anti-CGRP was purchased from Sigma and used at a dilution of 1:5000. CGRP is a neuropeptide found in pulmonary nerves and specifically expressed in PNE cells and NEBs of the airway epithelium. It is the most commonly used and ubiquitous immunohistochemical marker for cells of the PNE lineage.24Sunday ME Willett CG Patidar K Graham SA Modulation of oncogene and tumor suppressor gene expression in a hamster model of chronic lung injury with varying degrees of pulmonary neuroendocrine cell hyperplasia.Lab Invest. 1994; 70: 875-888PubMed Google Scholar, 28Wuenschell CW Sunday ME Singh G Minoo P Slavkin HD Warburton D Embryonic mouse lung epithelial progenitor cells co-express immunohistochemial markers of diverse mature cell lineages.J Histochem Cytochem. 1996; 44: 113-123Crossref PubMed Scopus (100) Google Scholar, 40Willett CG Shahsafei A Graham SA Sunday ME CD10/neutral endopeptidase inhibition augments pulmonary neuroendocrine cell hyperplasia in hamsters treated with diethylmitrosamine, and hyperoxia.Am J Respir Cell Mol Biol. 1999; 21: 13-20Crossref PubMed Scopus (11) Google Scholar, 41Borges M Linnoila RI van de Velde HJK Chen H Nelkin BD Mabry M Baylin SB Ball DW An achaete-scute holologue essential for neuroendocrine differentiation in the lung.Nature. 1997; 386: 852-855Crossref PubMed Scopus (341) Google Scholar Rabbit anti-CYP-2F2 was generated by Biosynthesis (Louisville, TX), using a synthetic peptide for amino acids 397–407. This polyclonal antibody was affinity purified using a peptide affinity column generated by the manufacture's directions (Pierce, Rockville, IL) and preadsorbed with spleen lysate. The affinity purified/preadsorbed CYP-2F2 antibody detects a single 53-kd protein on Western blots of both lung and liver lysates, two tissues known to express abundant CYP-2F2. No bands were detected on heart or spleen lysates (data not shown). This antibody was used at a dilution of 1:7000 for immunohistochemistry. Primary antibodies were diluted in PBS/0.5% BSA/5% normal goat serum. After an overnight incubation, the tissue was washed with PBS and incubated for 30 minutes at room temperature with biotinylated goat anti-rabbit secondary antibody (Sigma) diluted 1:2000 in PBS/0.5. BSA/5% normal goat serum. Tissue was washed with PBS and incubated at room temperature for 30 minutes with streptavidin-HRP (Zymed, South San Francisco, CA) diluted 1:250 in PBS. Antigen-antibody complexes were detected with diaminobenzidine (DAB). Analysis was carried out on an Olympus Provis AX70 microscope equipped with a Sony 9000 CCD video capture system linked to a Pentium processor PC running Image-Pro Plus (Media Cybernetics, Silver Spring, MD). Images from CGRP- and CCSP-immunostained tissue were acquired using similar microscope and camera settings. The specificity of the rabbit and goat anti-CCSP primary antibody42Cardoso WV Stewart LG Pinkerton KE Ji C Hook GE Singh G Katyal SL Thurlbeck WM Plopper CG Secretory product expression during Clara cell differentiation in the rabbit and rat.Am J Physiol. 1993; 264: L543-L552PubMed Google Scholar, 43Ten Have-Opbroek AA De Vries EC Clara cell differentiation in the mouse: ultrastructural morphology and cytochemistry for surfactant protein A and Clara cell 10 kD protein.Microsc Res Tech. 1993; 26: 400-411Crossref PubMed Scopus (34) Google Scholar was verified by immunohistochemical analysis of lung tissue from CCSP-null mice.44Stripp BR Lund J Mango GW Doyan KC Johnston C Juntenby K Nord M Whitsett JA Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals.Am J Physiol. 1996; 271: L656-L664PubMed Google Scholar, 45Reynolds SD Mango GW Gelein R Boe I-M Lund J Stripp BR Normal function and lack of fibronectin accumulation in kidneys of Clara cell secretory protein/uteroglobin deficient mice.Am J Kidney Dis. 1999; 33: 541-551Abstract Full Text Full Text PDF PubMed Google Scholar The absence of immunoreactivity on this tissue indicated that the rabbit and goat anti-CCSP antibodies are specific to CCSP and that there are no antigens within neuroepithelial cells that cross-react with this antibody. The rabbit anti-CGRP primary antibodies detect rare clusters of cells located primarily at branch points and walls opposite bifurcations and scattered single cells in untreated lung tissue. No reaction was observed on Clara cells or ciliated cells identified by morphology and position. Immunoreactivity was not observed in the absence of primary antibody. For dual immunohistochemistry, NEBs were identified by staining the middle of three adjacent serial sections for the PNE-specific antigen CGRP. Flanking sections were stained for the Clara cell marker CYP-2F2. Methods were as detailed above except that antibodies were diluted in PBS/3% BSA and amine-ethyl carbizol was used as the chromagen. Digital images of CGRP-stained NEB and NEB-associated CYP-2F2-positive cells were captured. The chromagen was removed with ethanol, and all sections were restained for the Clara cell marker CCSP as detailed above, using a goat anti-CCSP antibody (1:5000; G. Singh, University of Pittsburgh). Images of the same regions were overlaid by using Adobe Photoshop. Adjacent serial sections analyzed for proliferation were stained for CCSP or CGRP as detailed above. After immunohistochemistry, slides were washed overnight in PBS and dehydrated through ethanol. Dried slides were dipped in NTB-2 emulsion (Kodak; Rochester, NY), redried, and stored at 4°C for 1 week. Slides were developed by the manufacture's directions and counterstained with hematoxylin. Proliferative cells were defined as those containing five or more grains over the nucleus. All airway epithelial cells with a nuclear profile were counted for determination of the labeling index. Various labeling indices were calculated as follows: epithelial labeling index, 100 (number labeled cells/number airway epithelial cells); PNE labeling index, 100 (number labeled PNE/number PNE cells); % PNE, 100 (number PNE/number airway epithelial cells); PNE proliferative fraction, 100 (number labeled PNE/number labeled airway epithelial cells); and Clara cell proliferative fraction, 100 (number labeled Clara cells/number labeled airway epithelial cells). Paraffin sections were stained by the double indirect immunofluorescence method, using a combination of goat anti-CCSP and rabbit anti-CGRP. Sections were cleared with xylene, blocked with 20. normal horse serum in Tris-buffered saline for 45 minutes, and incubated overnight with primary antibody diluted 1:1000 in Tris-buffered saline/1% normal horse serum. Antigen antibody complexes were detected with Cy2-conjugated donkey anti-goat immunoglobulin (Jackson Immuno Research Laboratories, Inc., West Grove, PA) and Cy5-conjugated donkey anti-rabbit immunoglobulin (Jackson Immuno Research) diluted 1:100 in Tris-buffered saline/1% normal horse serum and incubated for 3 hours. All secondary antibodies are affinity purified and preadsorbed to minimize interspecies cross-reactivity. Sections were mounted in buffered glycerol and examined immediately using a Bethesda Research Laboratories (Bethesda, MD) confocal microscope equipped with argon and HeNe laser sources. Negative controls included incubation of sections with the inappropriate secondary antibody and omission of one or both primary antibodies from the staining protocol. The green and red images were overlaid with Adobe Photoshop. To define the spatial relationship between regenerating Clara cells and hyperplastic PNE cells, each population was localized in lung tissue of naphthalene-exposed mice by immunostaining adjacent serial tissue sections for CGRP and CCSP, respectively. Seventy-two hours after acute naphthalene exposure, there was a striking colocalization of CGRP- and CCSP-immunopositive (IP) cells, regardless of their anatomical location but including terminal bronchiole (Figure 1, A and B) and branch point (Figure 1, C–F) locations. Morphometric analysis indicated that 87% of CGRP-IP regions were associated with CCSP-IP cells. In all sections analyzed, CGRP domains represented a subset of CCSP-IP regions. NEBs, defined by clusters of CGRP-IP cells, were of consistent size throughout conducting airways of the repairing lung, with each NEB generally including 10–20 CGRP-IP cells that were in the plane of the section being analyzed. In contrast, the extent of the CCSP-positive region varied both in cell number and staining intensity (compare Figure 1, B and F, with D). Spatial colocalization of CGRP-IP and CCSP-IP cells indicates an intimate association between neuroepithelial bodies and nascent Clara cells and defines the branch point as a unique microenvironment for regeneration of the airway epithelium. A combination of immunostaining and autoradiographic detection of incorporated [3H]-thymidine was used to define progenitor-cell populations participating in repair of the naphthalene-injured airway epithelium at the 72-hour postexposure time point. Greater than 95% of labeled cells occurred within areas of clustered CGRP-IP cells (indicative of NEBs) that were associated with CCSP-IP cells. Within these regions, both CCSP- and CGRP-IP cell popu
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